PRIMER_OPT_TM=60.0 PRIMER_MIN_TM=58.0 PRIMER_MAX_TM=62.0 PRIMER_MAX_DIFF_TM=1.5
Forces the primers to have a G or C at the 3' end to improve binding specificity. primer3 input
Need to design a sequencing primer inside your PCR product? Use SEQUENCE_INTERNAL_OLIGO . PRIMER_OPT_TM=60
SEQUENCE_ID=example_seq_001 SEQUENCE_TEMPLATE=GACTGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGTAGCTAGCTAGCTAGCTAGCTAGCTAGCTAGCTAGCTAGCTAGCTAGCTAGCTAGCTAGCTAGCTAGCTAGCTAGCTAGCTAGC SEQUENCE_TARGET=50,100 PRIMER_OPT_SIZE=20 PRIMER_MIN_SIZE=18 PRIMER_MAX_SIZE=25 PRIMER_OPT_TM=60.0 PRIMER_MIN_TM=57.0 PRIMER_MAX_TM=63.0 = primer3 input
To get the best results, structure your input data properly:
The most commonly used interface is the Primer3 Input v. 0.4.0 (or v. 2.x) hosted by the University of Tartu. This web interface allows users to paste sequences and adjust parameters conveniently. Key Input Components